Cell Imaging Support—Getting Started

You should choose based upon the complexity of your labs’ analyses and whether you need transmitted (color) camera imaging or fluorescent (monochrome) camera imaging, or both. At routine/basic analyses, we offer the EVOS™ XL Core Imaging System for simple transmitted light imaging (typically used in cell culture facilities) or the FLoid™ Cell Imaging Station for dedicated three-color fluorescence imaging. The EVOS™ XL Imaging System differs from the EVOS™ XL Core Imaging System in that it has a larger monitor, a five objective turret (only four objectives with the EVOS™ XL Core instrument), can be networked, and can perform cell counting. 

 If you need more options for fluorescent wavelengths, cell counting, or time-lapse imaging, you may consider the EVOS™ FL Imaging System or the EVOS™ FL Auto Imaging System. The EVOS™ FL Auto Imaging System is our most versatile option, featuring automated imaging and more. It has both color and monochrome camera options, and can even be paired with our EVOS™ Onstage Incubator system.

Compare the capabilities of the EVOS™ Systems.

Yes, the Countess™ II FL instrument uses the same light cubes as the EVOS™ imaging systems. The Countess™ II instrument does not use light cubes.

All the EVOS™ imaging systems are inverted microscopes. For CC objectives, the coverslip must be face down, facing the objectives as the lenses have a short working distance suitable only for thin glass or plastic coverslips. LWD objectives are designed for viewing from the bottom of microplates, petri dishes, or culture flasks; the longer working distances of the lenses in these objectives accommodate thicker materials such as the plastic bottoms of various vessels. 

The FLoid™ Cell Imaging Station is a simple, easy-to-use transmitted-light and three-color fluorescence microscope. The three fluorescent colors, blue, green, and visible red (Texas Red™ dye) are set and cannot be changed to other filter sets. The magnification is also set with a 20x objective; this objective cannot be removed or changed.

On the other hand, the EVOS™ FL Imaging Systems are easy-to-use transmitted-light and fluorescence microscopes that allow the user access to the objective turret and light cube tray to permit multiple options for magnification and fluorescence detection. The EVOS™ FL Imaging Systems allow the use of 23 different light cubes and 18 different objectives (from 2x to 100x). 

• Plan Achromat objectives are perfect for general applications where color and focus have standard correction; these are suitable for samples requiring low magnification (2x to 4x). Plan Achromat objectives are recommended for basic brightfield microscopy and simple fluorescence detection.
• Plan Fluorite objectives provide the next level of improved resolution for brighter fluorescence signal and high contrast. These objectives are recommended for basic fluorescence imaging and brightfield microscopy at higher magnifications.
• Plan Apochromat provides the highest level of resolution, fluorescence brightness, contrast, and chromatic correction. If you are imaging very small structures and require high contrast and brightness, the Plan Apochromat objectives are the best option.

The GFP light cube (Ex = 470/22; Em = 510/42) has excitation centered at 470 nm covering 11 nm on each side for a total excitation range from 459 to 481 nm. Its emission is centered at 510 nm with 21 nm on each side for a total emission bandpass range from 489 to 531 nm. 

• The Qdot™ 525 light cube (Ex = 445/45; Em = 525/15) has excitation at 445 nm with 22.5 nm on each side for a total excitation range from 422.5 to 467.5. Its emission is centered at 525 nm with 7.5 nm on each side for a total emission bandpass range from 517.5 to 532.5. 
• The GFP light cube is best suited for fluorophores that are efficiently excited from 460 to 480 nm and emission with in the blue-green to green range. 
• The Qdot™ 525 light cube is better suited for exciting quantum dots at lower wavelengths (higher energy) and provides a narrower emission in the green range. 

Only the EVOS FL Auto Imaging System and EVOS FL Auto 2 Imaging System have an automatic cell counting function. The EVOS XL, EVOS FL, and EVOS FL Color imaging systems provide a manual cell counting tool that allows you to tag up to six different labels on the screen image.

We do offer an option for making custom vessel holders. Please contact your Sales Representative for initiating a custom order.

Wipe the vessel holder with paper towels or Kimwipes tissues dampened with 70% ethanol or 4,000 ppm hydrogen peroxide (H2O2).

Store the EVOS vessel holders flat and protected from any contact with heavy objects as they can be distorted. Once the holders are distorted and no longer lay flat on the stage, they cannot be used. Store them individually in their own lidded plastic containers or small sturdy boxes.

Yes you can, but we also offer the EVOS Onstage Incubator (Cat No. AMC1000) as a better option to control not just the temperature but also humidity and gas concentration for the EVOS FL Auto Imaging System. The EVOS Onstage Incubator cannot be used on the EVOS XL and FL imaging systems.

Yes, you can place the EVOS Light Shield box over the Tokai Hit Thermoplate and stage holder.

Wipe the thermoplate and vessel holder with paper towels or Kimwipes tissues dampened with 70% ethanol.

We recommend using long working distance (LWD) objectives as the glass heating plate is 0.5 mm thick, including the thickness of the culture plate or glass bottom dishes.

Contact Instrument Customer Services for any issues regarding the CellInsight™ systems and ArrayScan™ System. They can be reached at instrumentservices@thermofisher.com.

Some cell types accumulate phenol red, and this can pose a problem in the use of many fluorescent probes. Phenol red can quench visible-wavelength dyes and, although phenol red is non-fluorescent, various impurities may be fluorescent. We have many phenol red-free media to choose from. Our Live Cell Imaging Solution (HEPES-based) and our FluoroBrite™ DMEM have been optimized to be phenol red-free as well as to be non-autofluorescent.

Most media contain phenol red, which can quench fluorescent dyes in the visible wavelengths. Most media also contain autofluorescent components, such as riboflavin, which can reduce signal-to-background. We offer FluoroBrite™ DMEM and HEPES-based Live Cell Imaging Solution, which have been optimized for fluorescent imaging. We also offer a number of media without phenol red. But if none of these are reasonable options for your experiment, then we also offer BackDrop™ Background Suppressor ReadyProbes™ Reagent, which can be added to quench media autofluorescence.

As dyes are illuminated for imaging, they will fade, or “photobleach”, leading to unwanted dimming and lower detection efficiency over time. An antifade mounting medium can greatly reduce photobleaching. If you wish to label live cells, use of ProLong™ Live Antifade Reagent is helpful. If you wish to mount fixed cells after labeling, and then image immediately and then discard, SlowFade™ Diamond Antifade Mountant stays liquid but has good refractive index. If you wish to mount your sample and then archive the slides, ProLong™ Diamond Antifade Mountant will harden to a better refractive index and allow for archiving of the sample for up to several weeks, or even months. Unlike other antifade mounting media, these work well with fluorescent proteins for immediate viewing (archiving fluorescent proteins is not possible), and they are packaged with or without DAPI. More information on these can be found here.

This is possible for immediate viewing, but the sample cannot be archived with ProLong Live Antifade Reagent.

Yes. We recommend washing off the ProLong Live Antifade Reagent with pre-warmed buffer or media and proceeding to your standard fixation and permeabilization steps.

Yes, this is possible.

The evaporation of water is the “curing” process for Prolong, ProLong Gold, ProLong Diamond, and ProLong Glass antifade mountant. The evaporation of water causes the polymer to harden. There is no chemical reaction. Please note that there is no curing/hardening for any of the SlowFade reagents or ProLong Live products.

No. ProLong, ProLong Gold, ProLong Diamond, and ProLong Glass reagents are intended for use on fixed or fixed/permeabilized samples. Only ProLong Live Antifade Reagent (Cat Nos. P36974 and P36975) is intended for use with live cells.

We have not tested ProLong, ProLong Gold, ProLong Diamond, or ProLong Glass on DyLight dye-conjugated proteins or with other organic dyes, but it is likely that they will be compatible.

The concentration of NucBlue dye in this product is proprietary.

First, we recommend optimizing the amount of EdU and incubation time with the cells to ensure an adequate level of incorporation. Consider the cell's normal replication rate in optimizing the time they are exposed to EdU in the growth medium.

Second, we recommend optimizing the use of the Copper Protectant.

No. After 30 minutes, most of the buffer additive would have been oxidized.

Consider the normal replication rate for the cell of interest and culture the cells with multiple doses of EdU for one full cycle. For example, if cells divide every 24 hours, consider 3 or 4 additions of fresh EdU every 6 to 8 hrs and then fix and permeabilize within 2 to 3 hrs after the final addition. This helps to ensure a high level of EdU incorporation in the majority of the population that is replicating. This positive control provides proof that the click reaction and components are working properly.

The SuperBoost tyramide kits utilize poly-HRP labeled antibodies. This provides a greater number of horseradish peroxidase (HRP) molecules per antibody. The original kits used antibodies and streptavidin that were directly conjugated with HRP and thus, limited the number per antibody or streptavidin.

Yes. This involves the sequential application of the antibodies and the tyramides with a HRP-quenching step between antibodies using H2O2.

To limit background, we recommend performing a pre-blocking step with 3% H2O2 for 60 mins to inactivate endogenous peroxidases. To limit the localization of labeling, we recommend optimizing the final concentration of the primary and secondary antibodies and the dye-tyramide. You may also limit the incubation time of the dye-tyramide on the sample.

Compared to existing Alexa Fluor secondary antibodies, the new Alexa Fluor Plus secondary antibodies use a new proprietary dye chemistry coupled to highly cross-adsorbed antibodies, offering higher sensitivity and signal-to-noise ratios.

Because they are brighter, you would most likely be able to use a lower final concentration of the Alexa Fluor Plus secondary antibodies relative to the original Alexa Fluor secondary antibodies.

All Alexa Fluor Plus secondary antibodies are compatible with the SlowFade Diamond and ProLong Diamond Antifade Mountants.

We do not recommend doing this. The Image-iT Fixative Solution is 4% formaldehdye in PBS, so mixing it in directly with media would dilute the final concentration of fixative and this may result in less than optimal fixation. If you need to add fixative directly to the medium, consider using a fixative at a higher concentration such as formaldehyde at 16% (Cat. No. 28906) or at 37% (Cat. No. 9311).

Yes, it is alcohol-free.

Yes. Aldehyde-based fixation is compatible with other permeabilizing reagents, such as saponin.

The BODIPY FL dye is complexed to hydrophobic domains of the LDL; it is not covalently attached.

No. Exposure to detergents and surfactants can dissociate the BODIPY FL dye from the LDL.

If the BODIPY FL LDL is applied to live cells first and then fixed, the use of methanol or acetone fixation is not recommended. Exposure to alcohols, acetone, and other organic solvents can dissociate the BODIPY FL dye from the LDL.

The LDL in BODIPY FL LDL is not acetylated.

We do not recommend doing this as the oxidation process (such as oxidation with copper sulphate) may result in the degradation of the BODIPY FL dye.

pHrodo Red dye is covalently bound to the LDL.

pHrodo Red and Green dyes are pH-sensitive dyes. They should only be used on live cells to detect pH of cellular compartments. Upon fixation of the cells, the cell membrane is compromised and you are only measuring the pH of the buffer the samples are held in.

This product does not contain acetylated or oxidized LDL.

The Image-iT TMRM Reagent is a ready-to-use 1,000X concentrated stock solution of the TMRM dye. It contains 5 vials of 100 µM of TMRM in neat DMSO, with 100 µL volume in each vial. TMRM powder (Cat No. T668) is not included in this kit.

TMRM is a dynamic mitochondrial stain. There is no covalent attachment of TMRM to the mitochondria and because of this, it can move in and out of the mitochondria dependent upon changes in the membrane potential. The MitoTracker dyes on the other hand possess a reactive chloromethyl group that allows for their covalent attachment to mitochondria. As a result, once they accumulate within mitochondria that have a normal membrane potential, they are retained.

Yes, you may add TMRM to media with serum. Serum components may bind dye non-specifically so you may need a higher final working concentration for staining samples in media containing serum relative to staining the same cells in a non-serum media or buffer.