Lasers and suitable fluorochromes
If possible, avoid the traditional dyes like Fluorescein or FITC, use modern fluorochromes that are brighter and much more resistant to photobleaching. There are many brands/sources that work well.
|Laser||Fluorochrome (selected example)|
|405nm CW||DAPI, Hoechst, Calcofluor White, BFP|
|440 nm pulsed||CFP|
|WLL (470-670nm) pulsed||FITC*, Alexa Fluor 488… to Alexa Fluor 670, and other modern fluorochromes excited in this range, Cy3, Cy5, fluorescent protiens, ..|
FLUOROPHORE COMBINATION FOR MULTI-CHANNEL IMAGING
Choose dyes with well separated excitation and emission peaks. A combination of dyes with overlapping spectra (e.g., Dylight 488 and Cy3) is NOT recommended. When staining for DNA with DAPI or Hoechst dyes, remember that they have broad emission spectrum and will have an overlap with green-emitting dyes like FITC, Alexa Fluor 488. Sequential imaging will be necessary. There are many fluorescent proteins that can be used for single, double or more channels imaging
|DNA + 1 additional channel||DNA: DAPI or Hoechst (405 nm laser)|
2nd channel: Cy3/Alexa 568 or any dye with longer emission wavelength
Alternative 2nd channel: EGFP/Alexa Fluor488 – use sequential imaging
|DNA + 2 Additional channels||DNA: DAPI or Hoechst|
2nd channel: Cy3/Alexa 568
3rd channel Cy5/Alexa 647
ALternatively, 2nd channel: Alexa 488, 3rd channel Alexa 594 or 647. Use sequential imaging
|DNA + 3 additional channels||DNA: DAPI or Hoechst|
2nd channel: EGFP/Alexa 488
3rd channel : Texas Red or Alexa 594
4rd channel Cy5/Alexa 647
|two fluorescent proteins||EGFP (or mNeonGreen) and mCherry (or tdTomato) – there are many suitable combinations |
CFP and YFP
Best image quality is achieved if the mounting medium has the same refractive index as the immersion medium for the objective (Water – 1.33, Glycerol – 1.47, Oil – 1.518). To reduce photobleaching of the fluorophores, mounting media may contain anti-fade reagents. Some fluorophores are not compatible with certain antifade reagents. Consult the mounting media datasheet for compatibility with fluorescent dyes. Here are some standard mounting media and their refractive indices:.
- 75% Glycerol – 1.44
- Citifluor 1.463
- Vectashield – 1.457
- Mowiol – 1.49
- ProLong Gold (hardening version) – Fresh mounting: 1.39 Cured for 1 day: 1.40 Cured for 4 days: 1.44
- Prolong Glass, Citifluor High Refractive index media, 2,2-Thiodiethanol (Staudt et al., 2007) – 1.515
- Live samples, or samples mounted in a buffer are best imaged with a water immersion objective. Using oil immersion objectives for these samples is NOT recommended, resolution and signal strength will degrade rapidly when focusing deeper into the sample.
- For routine imaging of fixed, fluorescently stained samples, non-hardening glycerol-based mounting media are convenient and work reasonably well with oil immersion objectives as well as glycerol immersion objectives. You can make your own media (see links to recipes below) or use commercially available products.
- Best resolution for samples in glycerol mountants is achieved using the 63x/1.3 glycerol-immersion objective.
- For highest resolution imaging with oil immersion objectives, the samples should be mounted in a medium that has the same refractive index as glass and immersion oil (1.515). If it is a hardening medium, it may shrink during curing and distort 3D samples, however. The 2,2- thiodiethanol is a suitable high-index medium, but is not compatible with fluorescent proteins at the optimal (97%) concentration.
For more information, see this paper: Ravikumar S, Surekha R, Thavarajah R. Mounting media: An overview. J NTR Univ Health Sci 2014;3, Suppl S1:1-8
RECIPES FOR ANTI-FADE MOUNTING MEDIA
- Medium with n-propyl gallate. From Jackson Immunoresearch website www.jacksonimmuno.com: “In our experience n-propyl gallate added to mounting media reduces fading of fluorescence from many different fluorophores during fluorescence microscopy. The following is a simple recipe for making an anti-fade mounting medium containing n-propyl gallate.”
• Prepare a 10X PBS stock solution.
• Prepare a stock solution of 20%(w/v) n-propyl gallate (Sigma P3130) in dimethyl formamide or dimethyl sulfoxide. (Note: n-propyl gallate does not dissolve well in water-based solutions.)
• Thoroughly mix 1 part of 10X PBS with 9 parts of glycerol (ACS grade 99-100% purity) and slowly add 0.1 part 20% n-propyl gallate dropwise with rapid stirring.
Store the medium in aliquots at -20 C.
2. Medium with p-phenylenediamine (from CSHL Protocols http://cshprotocols.cshlp.org/content/2019/2/pdb.rec104265.full?rss=1):
|Reagent||Amount to add||Final concentration|
|Tris-HCl (1 M, pH 8.8)||0.8 mL||20 mM|
|Glycerol (100%)||36 mL||90%|
Mix the above ingredients together in a 50-mL conical tube. Adjust volume to 40 mL with H2O. Dissolve the solids by slowly bubbling nitrogen gas through a Pasteur pipette inserted deep into the mixture. Filter through a 0.8-µm filter at room temperature. Store at −20°C in 1- or 3-mL syringes devoid of air bubbles. Discard when dark brown. Handle this reagent with gloves; it causes stains when spilled.
Imaging Chambers, Slides and Plates
Microscope objectives are designed to view the sample through a thin glass window, 0.17 mm thick (this is the #1.5 coverglass). On the inverted microscope, the bottom of the sample chamber must be thin (0.17 mm glass) and optically clear. If your samples are in a standard plastic Petri dish or a regular multi-well culture plate, you will not be able to get the best image quality with the 10x objective, and you will not be able to focus at all with the other objectives.
Recommended types of samples:
- Slides with a 0.17 mm thick coverglass, sealed with nail polish or other suitable sealer that has dried
- Chambered slides where the culture chamber was removed after staining, suitable mounting medium and a coverglass was applied.
- Chambered slides or 35-mm Petri dishes with optically clear, thin bottom (e.g. Ibidi microslides, Greiner CELLview cell culture Slides)
- Imaging-type 96-well plates with optically clear, thin bottom – these MUST be specifically the type where the bottom is minimally recessed relative to the skirt of the plate. For example, Greiner Screenstar microplate (Item No.: 655866) or Eppendorf Cell Imaging Plate (Cat. # 003074103), or Ibidi µ-Plates should work well. CONSULT THE MIC STAFF IF YOU ARE NOT SURE WHICH PLATE YOU NEED.
- Chambered coverglass where the first and last chambers are very close to the ends – This creates a risk of damaging microscope objectives. This includes Sarsted x-well cell culture chamber, on coverglass, Labtek II chambered coverglass.
- Labtek I chambered coverglass – same problem as Labtek II, in adition, the coverglass is too thin. These WILL NOT FIT properly in the holder for live cell imaging.
- 95 well plates with deeply recessed bottom – They create a risk of damaging microscope objectives. The edge rows and columns CANNOT be imaged,
- If you use these plates or chambers, when the objective is in focus and you move to the well on the end or edge of the plate, the metal sample holder will hit the objective and damage it. You and your PI will be responsible for objective repair/replacement ($$$).