OPTICAL SECTIONING MICROSCOPY

Why are microscopy tools needed?

Most biological tissue is not see through (opaque) 

RI

Refractive index, the way light bends through other light sources

Light microscope uses

• resolution

• targeted contrast

• magnification

What are the problems with biological tissue?

• Opaque

• Does not transmit visible light well (lipid bilayers in the cell membrane)

• RI differences

• Not great for light microscopy

3 ways to solve opaque problem of biological tissue

1. use small samples (cell cultures)

2. Section/Cut Tissue to manageable size

3. Tissue Clearing

• cultured cells can transmit almost all light through them

• good for studying intracellular activity  

Why would smaller cell cultures not be the best? How could this problem be solved?

• Not the best for understanding how the cells are combining together or learning about the intact tissue  

• Trim tissue into ultra thin sections to get around the issue  

Optical Sectioning

Increases both contrast and resolution by eliminating out of focus light using rejection techniques

What does rejecting out of focus information improve?

Contrast and Resolution

Contrast

The ratio between the brightest and darkest regions of the image

Resolution

The ability to differentiate between 2 distinct points or objects

2 ways to create Optical Section

• Reject out-of-focus emission light (pinhole, structured illumination)

• Limit excitation volume

3 Methods to get an optical section

1. Confocal Microscopy

2. 2-Photon Microscopy

3. Lightsheet Microscopy

Large pinhole diameter =

Larger optical section

Smaller pinhole diameter =

Smaller optical section

How does confocal microscopy generate an optical section?

By physically rejecting out of focus light using an aperture placed in a conjugated focal plane

What does this image show?

Airy Pattern/Airy Disc

How the microscope sees a small point of light. Each individual fluorophore is seen as a spot in the center the rings surrounding are diffraction rings

What is a raster scanning device?

Confocal microscopes are a type of raster scanning device. Each pixel must be scanned in order to build an image. This is a slow process

Fluorescence signal is reduced as _____

Diameter is reduced

2 Photon microscopy

• Uses a different laser system to go through thicker tissue samples using NIR wavelengths  

• Good method for LIVE intact tissue  

• No aperture means significant improvement in photon collection

Tissue Clearing STEPS

1. Hydrogel embedding – passive diffusion

2. Polymerization – crosslink hydrogel under pressure and heat

3. Clearing – active electrophoretic field with detergent (SDS)

4. Labeling – IHC steps more effective with no lipid to permeabilize

5. Mounting/Imaging – using RI matching solution to hydrogel

LightSheet microscopy

• Generates an optical illumination section via cylindrical light shaping optics

• Fluorescence is detected perpendicular to illumination

• Sample can be moved in 4 dimensions

What is the main advantage of light sheet microscopy?

• camera based

• faster

• less photo damage, longer observation periods

• Does not collect out of focus fluorescence

Disadvantages of LightSheet

• Only illuminates a thing plane at a time

• Requires tissue clearing