Choosing right microscopy

• What questions are you trying to answer.
• What image data do i need.
• Availability
• How can you quantitate your images?

• Good for measuring cell behavior.
• No genetic encoded of flourecence needed.
• Less damaging than flourecence excitation

• Good for measuring molecular behavior.
• Are you looking at a thick or thin speciment?
• (Different  techniques based on depth)

• Living or fixed sample?
• (Rate of aquisition, cellular damage)

• advantages
• Low cost (no lasers)
• Easy to use

• disadvantages
• Lower signal to background
• (out of focus flourescence)
• More damaging to live cells

• Good for imaging the mitotic spindle
• Fixed Drosophila Cells
• Antibody staining

Computation processing of a Z stack of images

Epiflourescence microscopy with ony required hardware being a motorized Z axis stepper

Software required

• objects near the coverslip surface:
• Total Internal Reflection
• Flourescence (TIRF)

• <100 um thick (e.g. a cell)
• Pin hole microsopy (point or line scanning or spinning disk)

• >100 um thick (e.g. tissue):
• Two photon or array tomography (for larger tissue)

Single molucule Immunoprecipitation

Antibody is attached to surface is use to extract a molecule with flourescence tag.

Next gen-used to see colors

• <30um
• 1. Good for live cell imaging and fast acquisition

• <100 um
• 1. Good for fixed speciments
• (photodamage less of concern)
• 2. Good for combining photobleaching and imaging

• Travels perpendical to light axis
• no light damage
• Used for imaging embryos
• Low photodamage

Fast aquisition

• (100-500 um)
• 2 photon microscopy
• deeper into tissues

Physical sectioning and epiflourescence

Conventional - ~200-250 nm at best

• Structured Illumination- ~100 nm 
• Live Imaging

• STORM/PALM and STED- ~40 nm
• Most powerful for fixed sample

• Analysis for centrome
• looking at centrosomal proteins

• Imagej 
• Track molecule position
• speed
• behavior in motion