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5 Steps to Fixed-Cell Imaging
Improved fixed-cell imaging results
Pre-packaged reagent kits with optimized workflows combined with easy-to-use and powerful microscopes enable you to get the most from your fixed-cell imaging experiments. Below you can find products for fixation and permeabilization, cellular labeling, and image capture.
Get your free five-step guide for publication-quality cell imaging.
Fixed-cell imaging product selection guide
EVOS DAPI Light Cube (AMEP4650)
Excitation: 357/44 nm;
Emission: 447/60 nm
| Step | Application | Key tools for DAPI channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | ||
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS GFP Light Cube (AMEP4651)
Excitation: 470/22 nm;
Emission: 510/42 nm
| Step | Application | Key tools for GFP channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | Mitochondria | |
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Secondary antibodies | ||
| Streptavidin | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS RFP Light Cube (AMEP4652)
Excitation: 531/40 nm;
Emission: 593/40 nm
| Step | Application | Key tools for RFP channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | Mitochondria | |
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| Secondary antibodies | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS Red Light Cube (AMEP4655)
Excitation: 585/29 nm;
Emission: 624/40 nm
| Step | Application | Key tools for Texas Red channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | ||
| Cytoskeleton | ||
| Plasma membrane | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| Secondary antibodies | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS Cy5 Light Cube (AMEP4656)
Excitation: 628/40 nm;
Emission: 693/40 nm
| Step | Application | Key tools for Cy5 channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | Mitochondria | |
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS DAPI Light Cube (AMEP4650)
Excitation: 357/44 nm;
Emission: 447/60 nm
| Step | Application | Key tools for DAPI channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | ||
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS GFP Light Cube (AMEP4651)
Excitation: 470/22 nm;
Emission: 510/42 nm
| Step | Application | Key tools for GFP channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | Mitochondria | |
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Secondary antibodies | ||
| Streptavidin | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS RFP Light Cube (AMEP4652)
Excitation: 531/40 nm;
Emission: 593/40 nm
| Step | Application | Key tools for RFP channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | Mitochondria | |
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| Secondary antibodies | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS Red Light Cube (AMEP4655)
Excitation: 585/29 nm;
Emission: 624/40 nm
| Step | Application | Key tools for Texas Red channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | ||
| Cytoskeleton | ||
| Plasma membrane | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| Secondary antibodies | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
EVOS Cy5 Light Cube (AMEP4656)
Excitation: 628/40 nm;
Emission: 693/40 nm
| Step | Application | Key tools for Cy5 channel |
|---|---|---|
| Step 1. Fix, permeabilize, and block | Buffers |
|
| Step 2. Label | Mitochondria | |
| Cytoskeleton | ||
| Plasma membrane | ||
| Nucleus | ||
| Step 3. Detect | Antibody direct labeling kits | |
| Zenon kits | ||
| Streptavidin | ||
| SuperBoost TSA kits | ||
| Step 4. Protect and enhance | Signal enhancer | |
| Mountants and antifades | ||
| Step 5. Image | Imaging and analysis |
Follow these 5 steps to capture the best possible fixed-cell images
Step 1. Prepare
Prepare your cells for fluorescent labeling
To achieve optimal imaging quality, begin by setting up your study to spotlight proteins and cell structures of interest while keeping everything else out of the picture. Fixation and permeabilization prepare the cell samples for labeling—first by locking cellular structures, proteins, and nucleic acids in place, and then by making it possible for antibodies and fluorescent stains to permeate the interior of cells and label the targets of interest.
Fixation locks cellular structures in place.
Permeabilization removes cellular membrane lipids, enabling labeling and detection reagents to reach the interior of the cells.
Protein-based blocking agents help reduce nonspecific staining. Antibodies are able to displace the blocking proteins to form high-affinity interactions with their targets, while the remaining blocking proteins prevent low-affinity antibody interactions elsewhere in the sample.
After fixation and permeabilization, U2OS cells were stained with NucBlue Live Cell Stain and ActinGreen 488 ReadyProbes Reagent. Treatment A used methanol-based solution for fixation, and Treatment B used the formaldehyde-based Image-iT Fixation/Permeabilization Kit. The methanol-based fixation in A results in fragmentation of the actin cytoskeleton and disruption of the cells. The Image-iT Fixation/Permeabilization Kit provides optimal fixation conditions for most cell types.
| Product highlight | Description | Cat. No. |
|---|---|---|
| Image-iT Fixation/Permeabilization Kit |
| R37602 |
| BlockAid Blocking Solution |
| B10710 |
1.1 Culture conditions–Fixed cell imaging: 5 steps for publication-quality images
The first step in obtaining a good image is tissue preparation. For cultured cells, the cells must have good cell health and morphology, as well as good confluency. Healthy cells equal healthy data.
The first step in obtaining a good image is tissue preparation. For cultured cells, the cells must have good cell health and morphology, as well as good confluency. Healthy cells equal healthy data.
1.2 Fixation–Fixed cell imaging: 5 steps for publication-quality images
The next step of tissue preparation is fixation. Fixation refers to a chemical means of killing and preserving cells in a particular physiological state, and in many cases, to preserve morphology. Proper fixation equals preservation of target.
1.3 Permeabilization–Fixed cell imaging: 5 steps for publication-quality images
The next step is the permeabilization of the cells which is the key to opening intracellular compartments.
1.4 Blocking–Fixed cell imaging: 5 steps for publication-quality images
The next step after permeabilization is blocking, and there are a number of blocking techniques. Protein blocking equals specific antibody binding. Dye charge blocking means less non-specific binding.
1.5 Autofluorescence–Fixed cell imaging: 5 steps for publication-quality images
The last step in cell preparation is autofluorescence. Cells and tissue can have a certain degree of autofluorescence that can confuse the specific signal, and lower the signal-to-background. Overcoming autofluorescence means greater sensitivity.
Step 2. Label
Target cell structures and proteins of interest with organelle-selective dyes, stains, and primary antibodies
Labeling various targets with separate fluorescent colors allows you to visualize different structures or proteins within cells in the same sample. Ways to label your target include fluorescent dyes, immunolabeling, and fluorescent fusion proteins—all of which provide a means to selectively mark structures and molecules within the cell and allow you to see them more easily when you image.
Many fluorescence tools for cell biology are essentially fluorophores that have been modified in different ways or conjugated to various molecules to give them a certain function or allow them to bind to specific organelles or proteins. Through chemical modifications, a single fluorophore can be produced in various forms, each with a different specificity. For example, the green-fluorescent Invitrogen Alexa Fluor 488 dye molecule can be modified to target actin filaments, can be attached to an IgG for use in immunolabeling using our labeling kits, or can act as a whole-cell stain.
Product highlight
The Invitrogen portfolio offers more than 51,000 high-quality primary antibodies, with specificity to over 85% of the proteome. Some of these antibodies are attached directly to a broad range of intensely fluorescent markers and labels, including Invitrogen Alexa Fluor dyes.
A single fluorophore can be modified to carry out any number of labeling jobs, including functionalized forms for labeling cell structure components such as (A) actin, (B) tubulin, and (C) salt forms for whole-cell staining.
Cultured cells were prepared for staining using the Invitrogen Image-iT Fixation/Permeabilization Kit and were treated with Invitrogen BlockAid Blocking Solution. The sample was labeled with a primary antibody that recognizes mitochondria, followed by an Alexa Fluor 750 dye–conjugated secondary antibody (purple), Invitrogen NucBlue cell stain (blue), and Invitrogen ActinGreen 488 ReadyProbes Reagent (green). The image was captured on an Invitrogen EVOS FL Auto Imaging System.
2.1 Primary antibody choice–Fixed cell imaging: 5 steps for publication-quality images
After preparation, the second step to publishable images is to label the sample, usually involving primary antibodies to your specific targets of interest.
2.2 Primary antibody protocol optimization–Fixed cell imaging: 5 steps for publication-quality images
Every primary antibody must be optimized separately. There are many protocols available, and it is important to understand a “one size fits all” approach gives inferior results, as every antibody is slightly different.
Step 3. Detect
Fine-tune the fluorescence signal for observation
Detecting complex biological assemblies requires maximum clarity of fluorescence signals and separation of signals from background noise. Standard immunofluorescence labeling rarely provides the best signal-to-noise visibility. The difference between producing a good and a great publication-quality image requires fine-tuning your sample’s signal for peak specificity, definition, and amplification.
Fixed and permeabilized HeLa cells, treated using the reagents in the Image-iT Fixation/Permeabilization Kit, were incubated with an anti-tubulin primary antibody and an Alexa Fluor 488 goat anti–mouse IgG (H+L) secondary antibody. Cells were then incubated with an anti–ATP synthase subunit IF1 antibody and labeled with the reagents in the Alexa Fluor 594 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 594 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.
High- to medium-abundance protein targets
Secondary antibodies are used for the indirect detection of targets. While primary antibodies bind directly to the target, secondary antibodies bind indirectly by using the primary antibody as a bridge to the targeted biomolecule. Because multiple secondary antibody molecules can bind to a single primary antibody molecule, this methodology serves to amplify signals and increase sensitivity to maximize detection.
Explore secondary antibodies
For secondary detection, the primary antibodies (orange and yellow) bind to their respective epitopes and fluorophore-labeled secondary antibodies (purple and blue) have specificity for and bind to their respective primary antibodies.
Medium- to low-abundance protein targets
Streptavidin conjugates can increase the number of fluorophores that label your target, and boost their signals. Streptavidin-based amplification techniques are widely used in fluorescence imaging for improved sensitivity of detection with primary and secondary antibodies.
Find out more about streptavidin signal amplification for imaging
Antibodies labeled using fluorophore–streptavidin conjugates can boost low signals in your labeling experiments because more fluorophores can bind to each antibody molecule, amplifying the signals.
Low-abundance protein targets
For low-abundance protein targets that are not detectable by conventional means, tyramide signal amplification (TSA, PerkinElmer) provides sensitive detection without compromising resolution. TSA technology employs an enzyme that releases reactive dyes in the presence of hydrogen peroxide to bring targets out of the background with definition and clarity.
Learn more about imaging low-abundance targets with TSA
Click image to enlarge
Tyramide signal amplification provides sensitive detection by causing localized high-density deposition of fluorescent labels around epitopes, resulting in superior sensitivity without compromising resolution.
Product highlight
Our Alexa Fluor Plus secondary antibodies uses proprietary plus dye chemistry to offer up to 4.2 times higher signal-to-noise ratios compared to our Alexa Fluor formulation, providing higher sensitivity to detect low-abundance targets.
The Invitrogen Tyramide SuperBoost kits uses the Invitrogen SuperBoost technology to provide the most sensitive fluorescence imaging detection method for low-abundance protein targets. Offering sensitivity 10-200 times that of standard immunochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH) methods, SuperBoost kits are designed for superior signal amplification, definition, and clarity needed for high-resolution imaging. Combining the brightness of Alexa Fluor dyes with trusted poly-HRP-mediated tyramide signal amplification, the SuperBoost reagent generates sensitivity typically 2-10 times above that of standard treatments, including reagents from other suppliers.
3.1 Secondary antibody choice–Fixed cell imaging: 5 steps for publication-quality images
Step three of the five steps in making publishable images is to detect the label. That is, to detect with a secondary antibody, for instance, or an amplification technique, as well as to determine what controls to use. Your options are discussed.
3.2 Secondary antibody optimization–Fixed cell imaging: 5 steps for publication-quality images
Secondary antibody detection protocols also need to be optimized for each primary antibody used.
3.3 Amplification techniques–Fixed cell imaging: 5 steps for publication-quality images
If the signal is not strong enough using standard secondary detection schemes, you can increase the signal using amplification techniques. This is particularly important for low-expressing antigens, or rare-cell detection in samples.
3.4 Controls–Fixed cell imaging: 5 steps for publication-quality images
Researchers should conduct all necessary controls to rule out the possibility of non-specific binding or non-specific signal. Types of controls are described. Proper controls will boost your confidence in your final results.
3.5 Dye choice and special concerns–Fixed cell imaging: 5 steps for publication-quality images
There are many different dyes spanning the visible, far-red, and infrared wavelengths.Considerations for making the right choices for your experiment are presented.
Step 4. Protect
Protect fluorescent signal from photobleaching and enhance image quality with the best resolution
Fluorophores are ideal for high-quality cell imaging but are inevitably prone to photobleaching, a photochemical degradation and fading of fluorescence signals. Any reduction in photosensitivity can skew your data and yield false results. Antifade mountants are designed to protect the photostability of fluorophores and maintain image integrity for weeks to months.
Product highlight
Invitrogen ProLong Glass antifade mountants are designed to provide unparalleled antifade protection across the entire visible and near-infrared spectrum. With a refractive index of 1.52, ProLong Glass mountants can be used with many fluorescent dyes on virtually any cell or tissue sample ranging from 0.1 µm to 150 µm for bright, high-resolution Z-stack, 3D, and 2D images. They also work particularly well with oil-immersion lenses and confocal microscopes due to decreased distortion of the images.
Key benefits of ProLong antifade mountants for fixed cells include:
- Inhibit photobleaching across the spectrum, even after prolonged storage
- Low background across the spectrum
- Hard-setting mountants
- Available with or without DAPI
- Ready-to-use benchtop formulations
- Choice of refractive index based on your needs: 1.52 for ProLong Glass reagent and 1.47 for ProLong Diamond and ProLong Gold reagents
A 60-second time-lapse showing the enhanced resistance to photobleaching afforded by ProLong antifade mountants. Fixed HeLa cells were labeled with fluorescein phalloidin and mounted in ProLong Glass reagent, ProLong Diamond reagent, ProLong Gold reagent, or 50% PBS/glycerol. Images were acquired at 12-second intervals using a 20x objective with continuous illumination from a standard 100-watt Hg-arc lamp.
Featured Scientific Poster
Antifade mounting media to help improve image quality in 3D biological samples
4.1 Mounting media–Fixed cell imaging: 5 steps for publication-quality images
Using the right mounting media can impact your experiment. Be sure to choose the right type of mountant for your set-up.
4.2 Photobleaching and antifades–Fixed cell imaging: 5 steps for publication-quality images
What is photobleaching and how can you prevent it from destroying your sample? Options for antifades are discussed.
Step 5. Image
Capture imaging discoveries with maximum clarity and definition
Optimize this step to capture research discoveries with maximum clarity and definition. In today’s competitive scientific environment, generating publication-quality images is critical to your success. To capture top-quality images, you need an imaging platform with top-of-the-line imaging components, including:
- High-quality cameras and optics to capture high-resolution images
- LED illumination to produce superior signal-to-noise ratios
- Easy-to-use image capture and processing software for ready-to-publish images
- The ability to analyze images for extensive quantitative and statistical analysis
Product highlight
Choose easy-to-use, modular systems that can adjust to your experimental needs. We offer imaging systems that can be customized with a variety of LED light cubes, vessel holders, and objectives. There are more than 14 Invitrogen EVOS LED light cubes to choose from, covering a broad range of fluorescence excitation and emission.
Need to acquire more quantitative data while increasing sample throughput? The CellInsight CX5 High Content Analysis platform provides automated image capture with simultaneous data analysis, allowing you to analyze up to half a million phenotypic cell measurement in under 5 minutes.
Compare imaging systems below to find the perfect fit.
Get more information on our entire line of microscopy cell imaging systems
Perfect for quick fluorescence visualization
| Basic transmitted light-digital inverted system | Advanced transmitted-light digital inverted system | Basic fluorescence system | Advanced fluorescence system | Fully automated fluorescence system |
|---|---|---|---|---|
 EVOS XL Core Perfect for cell culture and routine cell maintenance |  EVOS XL Perfect for more advanced colormetric assays |  EVOS FLoid Perfect for quick fluorescence visualization |
|
|
Get more information on our entire line of high-content analysis cell imaging systems
| 5-channel LED system | 7-channel LED confocal system | 7-channel laser confocal system |
|---|---|---|
 CellInsight CX5 Perfect for labs looking for a compact and affordable system to increase scale See reagents |  CellInsight CX7 Perfect for labs looking for additional choices of imaging mode See reagents |  CellInsight CX7 LZR Perfect for labs looking for advanced performance in sensitivity and speed See reagents |
Get more information on our entire line of microscopy cell imaging systems
Perfect for quick fluorescence visualization
| Basic transmitted light-digital inverted system | Advanced transmitted-light digital inverted system | Basic fluorescence system | Advanced fluorescence system | Fully automated fluorescence system |
|---|---|---|---|---|
EVOS XL Core Perfect for cell culture and routine cell maintenance | EVOS XL Perfect for more advanced colormetric assays | EVOS FLoid Perfect for quick fluorescence visualization |
|
|
Get more information on our entire line of high-content analysis cell imaging systems
| 5-channel LED system | 7-channel LED confocal system | 7-channel laser confocal system |
|---|---|---|
CellInsight CX5 Perfect for labs looking for a compact and affordable system to increase scale See reagents | CellInsight CX7 Perfect for labs looking for additional choices of imaging mode See reagents | CellInsight CX7 LZR Perfect for labs looking for advanced performance in sensitivity and speed See reagents |
5.1 Imaging platforms-hardware–Fixed cell imaging: 5 steps for publication-quality images
The fifth step of the process is the actual imaging. To capture top-quality images, you need an imaging platform with top-of-the-line imaging capabilities. Here we review considerations for getting the best image.
5.2 Imaging platforms-software–Fixed cell imaging: 5 steps for publication-quality images
Taking images on a microscope usually entails having some type of imaging software that aids in taking the image and assists in combining differing colors into one. There are some very important aspects to keep in mind to get a publishable image.
5.3 Image capture with EVOS FL Auto 2.0–Fixed cell imaging: 5 steps for publication-quality images
Here the advantages of using the EVOS FL Auto 2.0 imaging system to capture your images are discussed.
5.4 Image analysis with Celleste software–Fixed cell imaging: 5 steps for publication-quality images
The functionality of the Celleste software is reviewed and considerations for processing the image in different software programs are described.
5.5 Ethical considerations–Fixed cell imaging: 5 steps for publication-quality images
Ethical imaging means trustworthy data, and thus, publishable data. How to treat your samples and data to preserve data integrity is presented.
Step 1. Prepare
Prepare your cells for fluorescent labeling
To achieve optimal imaging quality, begin by setting up your study to spotlight proteins and cell structures of interest while keeping everything else out of the picture. Fixation and permeabilization prepare the cell samples for labeling—first by locking cellular structures, proteins, and nucleic acids in place, and then by making it possible for antibodies and fluorescent stains to permeate the interior of cells and label the targets of interest.
Fixation locks cellular structures in place.
Permeabilization removes cellular membrane lipids, enabling labeling and detection reagents to reach the interior of the cells.
Protein-based blocking agents help reduce nonspecific staining. Antibodies are able to displace the blocking proteins to form high-affinity interactions with their targets, while the remaining blocking proteins prevent low-affinity antibody interactions elsewhere in the sample.
After fixation and permeabilization, U2OS cells were stained with NucBlue Live Cell Stain and ActinGreen 488 ReadyProbes Reagent. Treatment A used methanol-based solution for fixation, and Treatment B used the formaldehyde-based Image-iT Fixation/Permeabilization Kit. The methanol-based fixation in A results in fragmentation of the actin cytoskeleton and disruption of the cells. The Image-iT Fixation/Permeabilization Kit provides optimal fixation conditions for most cell types.
| Product highlight | Description | Cat. No. |
|---|---|---|
| Image-iT Fixation/Permeabilization Kit |
| R37602 |
| BlockAid Blocking Solution |
| B10710 |
1.1 Culture conditions–Fixed cell imaging: 5 steps for publication-quality images
The first step in obtaining a good image is tissue preparation. For cultured cells, the cells must have good cell health and morphology, as well as good confluency. Healthy cells equal healthy data.
The first step in obtaining a good image is tissue preparation. For cultured cells, the cells must have good cell health and morphology, as well as good confluency. Healthy cells equal healthy data.
1.2 Fixation–Fixed cell imaging: 5 steps for publication-quality images
The next step of tissue preparation is fixation. Fixation refers to a chemical means of killing and preserving cells in a particular physiological state, and in many cases, to preserve morphology. Proper fixation equals preservation of target.
1.3 Permeabilization–Fixed cell imaging: 5 steps for publication-quality images
The next step is the permeabilization of the cells which is the key to opening intracellular compartments.
1.4 Blocking–Fixed cell imaging: 5 steps for publication-quality images
The next step after permeabilization is blocking, and there are a number of blocking techniques. Protein blocking equals specific antibody binding. Dye charge blocking means less non-specific binding.
1.5 Autofluorescence–Fixed cell imaging: 5 steps for publication-quality images
The last step in cell preparation is autofluorescence. Cells and tissue can have a certain degree of autofluorescence that can confuse the specific signal, and lower the signal-to-background. Overcoming autofluorescence means greater sensitivity.
Step 2. Label
Target cell structures and proteins of interest with organelle-selective dyes, stains, and primary antibodies
Labeling various targets with separate fluorescent colors allows you to visualize different structures or proteins within cells in the same sample. Ways to label your target include fluorescent dyes, immunolabeling, and fluorescent fusion proteins—all of which provide a means to selectively mark structures and molecules within the cell and allow you to see them more easily when you image.
Many fluorescence tools for cell biology are essentially fluorophores that have been modified in different ways or conjugated to various molecules to give them a certain function or allow them to bind to specific organelles or proteins. Through chemical modifications, a single fluorophore can be produced in various forms, each with a different specificity. For example, the green-fluorescent Invitrogen Alexa Fluor 488 dye molecule can be modified to target actin filaments, can be attached to an IgG for use in immunolabeling using our labeling kits, or can act as a whole-cell stain.
Product highlight
The Invitrogen portfolio offers more than 51,000 high-quality primary antibodies, with specificity to over 85% of the proteome. Some of these antibodies are attached directly to a broad range of intensely fluorescent markers and labels, including Invitrogen Alexa Fluor dyes.
A single fluorophore can be modified to carry out any number of labeling jobs, including functionalized forms for labeling cell structure components such as (A) actin, (B) tubulin, and (C) salt forms for whole-cell staining.
Cultured cells were prepared for staining using the Invitrogen Image-iT Fixation/Permeabilization Kit and were treated with Invitrogen BlockAid Blocking Solution. The sample was labeled with a primary antibody that recognizes mitochondria, followed by an Alexa Fluor 750 dye–conjugated secondary antibody (purple), Invitrogen NucBlue cell stain (blue), and Invitrogen ActinGreen 488 ReadyProbes Reagent (green). The image was captured on an Invitrogen EVOS FL Auto Imaging System.
2.1 Primary antibody choice–Fixed cell imaging: 5 steps for publication-quality images
After preparation, the second step to publishable images is to label the sample, usually involving primary antibodies to your specific targets of interest.
2.2 Primary antibody protocol optimization–Fixed cell imaging: 5 steps for publication-quality images
Every primary antibody must be optimized separately. There are many protocols available, and it is important to understand a “one size fits all” approach gives inferior results, as every antibody is slightly different.
Step 3. Detect
Fine-tune the fluorescence signal for observation
Detecting complex biological assemblies requires maximum clarity of fluorescence signals and separation of signals from background noise. Standard immunofluorescence labeling rarely provides the best signal-to-noise visibility. The difference between producing a good and a great publication-quality image requires fine-tuning your sample’s signal for peak specificity, definition, and amplification.
Fixed and permeabilized HeLa cells, treated using the reagents in the Image-iT Fixation/Permeabilization Kit, were incubated with an anti-tubulin primary antibody and an Alexa Fluor 488 goat anti–mouse IgG (H+L) secondary antibody. Cells were then incubated with an anti–ATP synthase subunit IF1 antibody and labeled with the reagents in the Alexa Fluor 594 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 594 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.
High- to medium-abundance protein targets
Secondary antibodies are used for the indirect detection of targets. While primary antibodies bind directly to the target, secondary antibodies bind indirectly by using the primary antibody as a bridge to the targeted biomolecule. Because multiple secondary antibody molecules can bind to a single primary antibody molecule, this methodology serves to amplify signals and increase sensitivity to maximize detection.
Explore secondary antibodies
For secondary detection, the primary antibodies (orange and yellow) bind to their respective epitopes and fluorophore-labeled secondary antibodies (purple and blue) have specificity for and bind to their respective primary antibodies.
Medium- to low-abundance protein targets
Streptavidin conjugates can increase the number of fluorophores that label your target, and boost their signals. Streptavidin-based amplification techniques are widely used in fluorescence imaging for improved sensitivity of detection with primary and secondary antibodies.
Find out more about streptavidin signal amplification for imaging
Antibodies labeled using fluorophore–streptavidin conjugates can boost low signals in your labeling experiments because more fluorophores can bind to each antibody molecule, amplifying the signals.
Low-abundance protein targets
For low-abundance protein targets that are not detectable by conventional means, tyramide signal amplification (TSA, PerkinElmer) provides sensitive detection without compromising resolution. TSA technology employs an enzyme that releases reactive dyes in the presence of hydrogen peroxide to bring targets out of the background with definition and clarity.
Learn more about imaging low-abundance targets with TSA
Click image to enlarge
Tyramide signal amplification provides sensitive detection by causing localized high-density deposition of fluorescent labels around epitopes, resulting in superior sensitivity without compromising resolution.
Product highlight
Our Alexa Fluor Plus secondary antibodies uses proprietary plus dye chemistry to offer up to 4.2 times higher signal-to-noise ratios compared to our Alexa Fluor formulation, providing higher sensitivity to detect low-abundance targets.
The Invitrogen Tyramide SuperBoost kits uses the Invitrogen SuperBoost technology to provide the most sensitive fluorescence imaging detection method for low-abundance protein targets. Offering sensitivity 10-200 times that of standard immunochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH) methods, SuperBoost kits are designed for superior signal amplification, definition, and clarity needed for high-resolution imaging. Combining the brightness of Alexa Fluor dyes with trusted poly-HRP-mediated tyramide signal amplification, the SuperBoost reagent generates sensitivity typically 2-10 times above that of standard treatments, including reagents from other suppliers.
3.1 Secondary antibody choice–Fixed cell imaging: 5 steps for publication-quality images
Step three of the five steps in making publishable images is to detect the label. That is, to detect with a secondary antibody, for instance, or an amplification technique, as well as to determine what controls to use. Your options are discussed.
3.2 Secondary antibody optimization–Fixed cell imaging: 5 steps for publication-quality images
Secondary antibody detection protocols also need to be optimized for each primary antibody used.
3.3 Amplification techniques–Fixed cell imaging: 5 steps for publication-quality images
If the signal is not strong enough using standard secondary detection schemes, you can increase the signal using amplification techniques. This is particularly important for low-expressing antigens, or rare-cell detection in samples.
3.4 Controls–Fixed cell imaging: 5 steps for publication-quality images
Researchers should conduct all necessary controls to rule out the possibility of non-specific binding or non-specific signal. Types of controls are described. Proper controls will boost your confidence in your final results.
3.5 Dye choice and special concerns–Fixed cell imaging: 5 steps for publication-quality images
There are many different dyes spanning the visible, far-red, and infrared wavelengths.Considerations for making the right choices for your experiment are presented.
Step 4. Protect
Protect fluorescent signal from photobleaching and enhance image quality with the best resolution
Fluorophores are ideal for high-quality cell imaging but are inevitably prone to photobleaching, a photochemical degradation and fading of fluorescence signals. Any reduction in photosensitivity can skew your data and yield false results. Antifade mountants are designed to protect the photostability of fluorophores and maintain image integrity for weeks to months.
Product highlight
Invitrogen ProLong Glass antifade mountants are designed to provide unparalleled antifade protection across the entire visible and near-infrared spectrum. With a refractive index of 1.52, ProLong Glass mountants can be used with many fluorescent dyes on virtually any cell or tissue sample ranging from 0.1 µm to 150 µm for bright, high-resolution Z-stack, 3D, and 2D images. They also work particularly well with oil-immersion lenses and confocal microscopes due to decreased distortion of the images.
Key benefits of ProLong antifade mountants for fixed cells include:
- Inhibit photobleaching across the spectrum, even after prolonged storage
- Low background across the spectrum
- Hard-setting mountants
- Available with or without DAPI
- Ready-to-use benchtop formulations
- Choice of refractive index based on your needs: 1.52 for ProLong Glass reagent and 1.47 for ProLong Diamond and ProLong Gold reagents
A 60-second time-lapse showing the enhanced resistance to photobleaching afforded by ProLong antifade mountants. Fixed HeLa cells were labeled with fluorescein phalloidin and mounted in ProLong Glass reagent, ProLong Diamond reagent, ProLong Gold reagent, or 50% PBS/glycerol. Images were acquired at 12-second intervals using a 20x objective with continuous illumination from a standard 100-watt Hg-arc lamp.
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Antifade mounting media to help improve image quality in 3D biological samples
4.1 Mounting media–Fixed cell imaging: 5 steps for publication-quality images
Using the right mounting media can impact your experiment. Be sure to choose the right type of mountant for your set-up.
4.2 Photobleaching and antifades–Fixed cell imaging: 5 steps for publication-quality images
What is photobleaching and how can you prevent it from destroying your sample? Options for antifades are discussed.
Step 5. Image
Capture imaging discoveries with maximum clarity and definition
Optimize this step to capture research discoveries with maximum clarity and definition. In today’s competitive scientific environment, generating publication-quality images is critical to your success. To capture top-quality images, you need an imaging platform with top-of-the-line imaging components, including:
- High-quality cameras and optics to capture high-resolution images
- LED illumination to produce superior signal-to-noise ratios
- Easy-to-use image capture and processing software for ready-to-publish images
- The ability to analyze images for extensive quantitative and statistical analysis
Product highlight
Choose easy-to-use, modular systems that can adjust to your experimental needs. We offer imaging systems that can be customized with a variety of LED light cubes, vessel holders, and objectives. There are more than 14 Invitrogen EVOS LED light cubes to choose from, covering a broad range of fluorescence excitation and emission.
Need to acquire more quantitative data while increasing sample throughput? The CellInsight CX5 High Content Analysis platform provides automated image capture with simultaneous data analysis, allowing you to analyze up to half a million phenotypic cell measurement in under 5 minutes.
Compare imaging systems below to find the perfect fit.
Get more information on our entire line of microscopy cell imaging systems
Perfect for quick fluorescence visualization
| Basic transmitted light-digital inverted system | Advanced transmitted-light digital inverted system | Basic fluorescence system | Advanced fluorescence system | Fully automated fluorescence system |
|---|---|---|---|---|
EVOS XL Core Perfect for cell culture and routine cell maintenance | EVOS XL Perfect for more advanced colormetric assays | EVOS FLoid Perfect for quick fluorescence visualization |
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Get more information on our entire line of high-content analysis cell imaging systems
| 5-channel LED system | 7-channel LED confocal system | 7-channel laser confocal system |
|---|---|---|
CellInsight CX5 Perfect for labs looking for a compact and affordable system to increase scale See reagents | CellInsight CX7 Perfect for labs looking for additional choices of imaging mode See reagents | CellInsight CX7 LZR Perfect for labs looking for advanced performance in sensitivity and speed See reagents |
Get more information on our entire line of microscopy cell imaging systems
Perfect for quick fluorescence visualization
| Basic transmitted light-digital inverted system | Advanced transmitted-light digital inverted system | Basic fluorescence system | Advanced fluorescence system | Fully automated fluorescence system |
|---|---|---|---|---|
EVOS XL Core Perfect for cell culture and routine cell maintenance | EVOS XL Perfect for more advanced colormetric assays | EVOS FLoid Perfect for quick fluorescence visualization |
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Get more information on our entire line of high-content analysis cell imaging systems
| 5-channel LED system | 7-channel LED confocal system | 7-channel laser confocal system |
|---|---|---|
CellInsight CX5 Perfect for labs looking for a compact and affordable system to increase scale See reagents | CellInsight CX7 Perfect for labs looking for additional choices of imaging mode See reagents | CellInsight CX7 LZR Perfect for labs looking for advanced performance in sensitivity and speed See reagents |
5.1 Imaging platforms-hardware–Fixed cell imaging: 5 steps for publication-quality images
The fifth step of the process is the actual imaging. To capture top-quality images, you need an imaging platform with top-of-the-line imaging capabilities. Here we review considerations for getting the best image.
5.2 Imaging platforms-software–Fixed cell imaging: 5 steps for publication-quality images
Taking images on a microscope usually entails having some type of imaging software that aids in taking the image and assists in combining differing colors into one. There are some very important aspects to keep in mind to get a publishable image.
5.3 Image capture with EVOS FL Auto 2.0–Fixed cell imaging: 5 steps for publication-quality images
Here the advantages of using the EVOS FL Auto 2.0 imaging system to capture your images are discussed.
5.4 Image analysis with Celleste software–Fixed cell imaging: 5 steps for publication-quality images
The functionality of the Celleste software is reviewed and considerations for processing the image in different software programs are described.
5.5 Ethical considerations–Fixed cell imaging: 5 steps for publication-quality images
Ethical imaging means trustworthy data, and thus, publishable data. How to treat your samples and data to preserve data integrity is presented.
Color your way through 30 fantastic illustrations inspired by actual cell images submitted by researchers around the world. Don’t wait! Download the free coloring book that helps inspire your creative scientific expression.
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