MBI | Tips & Tricks
1. Coverslip Thickness
Generally, most of the immersion lens are calibrated for coverslips with a thickness of 0.17mm(which is falls in the range of #1.5 coverslip).
But some brands are always better than the others, check your coverslip thickness if necessary.
2. Coverslip cleaning protocols
2.a Preparation of Squeaky-Clean Coverslips*
1) Place coverslips one at a time in a 500 ml glass beaker filled halfway with hot tap water containing ~5 ml of Versa Clean detergent, taking care to separate coverslips that are stuck together. Sonicate coverslips in hot tap water containing Versa Clean for 45 min in a water bath sonicator.
2) Rinse coverslips 10 times by swirling with hot tap water. Sonicate in hot tap water for 30 min.
3) Rinse coverslips 10 times by swirling with double distilled water. Sonicate in double distilled water for 30 min.
4) Rinse coverslips 3 times by swirling with 1 mM EDTA. Sonicate in 1 mM EDTA for 30 min.
5) Rinse coverslips 3 times by swirling with 70 % ethanol. Sonicate in 70% ethanol for 30 min.
6) Rinse coverslips 3 times by swirling with absolute ethanol. Sonicate in absolute ethanol for 30 min.
7) Rinse coverslips once with absolute ethanol, transfer them to a 500 ml screw cap jar, cover them with absolute ethanol, and store at room temperature until use.
*=As published in...
Waterman-Storer, C.M. (In press) Microtubule/organelle motility assays. In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. John Wiley, NY.
3. "The Specimen is an integral part of any Microscope system and its optical characteristics directly affect the performance of the microscope."
P.C. Cheng, Chpt 21, Handbook of Biological Confocal Microscope.
For mammalian cell culture/tissue slice, the optical property, to which all microscope users should pay special attention to, is autofluorescence. Do read up a bit on possible autofluorescence of your sample before purchasing dyes.
4. Long Term Imaging Water?
Zeiss Immersol W
Same Refractive Index as water
Will not dry out in 5 mins
5. Sealing the slides.
A quick way to test the usability for sealing slides.
Buy different brands of nail hardener.Fix a coverslip on a slide with nail hardener, let dry overnight, and put under water next day. The one prep dissolving latest is the best one.
Expensive/good quality ones are wise to buy as the cheap ones seems to have a solvent that will destroy the fluorochrome or even the whole cell.
Usually have some form of plasticizer or other additive that gives autofluorescence. Can be replaced by clear lacquer(漆)from hardware, all ingredients are clearly identified in the hardware product.
For live cell, use "valap", a 1:1:1 hombrew mixture of vaseline, lanolin and parrafin.
Why use colourless? I usually use something nice and bright, so that you can see at a glance that the slide is sealed. It also gives you immediate warning when you are getting close to the edge of the coverslip. Mind you I do sometimes get funny looks when people see blue and orange nail polish bottles on my desk!
6.Test Beads， Fiducials
Multi Color/Single Color beads of various size are a must to have for Imaging Labs.
7. Live Cell Imaging Chambers
OptiCell, BioCrystal Ltd.
Respiratory Active Membrae,75um poly
BioFolie25 In Vitro System and Services
Autoclave Friendly Gas Permeable Membrane, 25um Thick RI=1.35
Imaging Processing Softwares:
ImageJ(Powerful freeware, even more powerful if used with plugin)
Confocal Assistant (Freeware, windows)
Imaris(Commercial Software from BitPlane, an Andor sub-company)
SlideBook (Commercial Software from 3i)
Huygens (Commercial Software from Scientific Volume Imaging, Deconvolution)
Confocal List Server
An excellent international forum where confocal users discuss confocal imaging
Nikon A1R Si Offline Image Viewing Software
A very useful educational site for understanding all aspects of light microscopy, confocal and digital imaging http://micro.magnet.fsu.edu/primer/index.html
Fluorescence and Confocal Specimen Preparation http://bama.ua.edu/~hsmithso/class/bsc_656/websites/sample_fluorescence.html
Microscopy Basics (Systematic Knowledge Base)
Online Fluorophore Spectra Mixing
Confocal Microscopy for Biologists, Alan R. Hibbs, Springer
Handbook of Biological Confocal Microscopy, James B. Pawley, Springer
Other Research and Core Facility Sites:
Microscopy Page of Prof. Colin Sheppard
Microscope Manufacturer Links:
Former Bio-Rad Cell Science Division
Accessories and Other Related Suppliers:
Andor Technology (CCDs , Spinning Disk System)
Applied Precision (Motorize Stage, Fluorensence Microscopy System)
MatTek (Glass Bottom Dishes For Live Cell Imaging)
Molecular Probes (Dyes Database)
OKOLabs(Live Imaging Fluorensence Automated System)
Semrock(Hard Coating Filters)
Warner Instrument(Perfusion Chambers)