Optogenetics and Light Sheet Fluorescence Microscopy

Optogenetics

Science of using light to control neurons and influence thoughts, emotions, and behaviors.

Opsins

Proteins that convert light into electrical signals.

Excitation

Light activates neurons (gain of function).

Inhibition

Light deactivates neurons (loss of function).

Fiber Optics

Light penetrates the brain via optical fibers, reaching deeper regions.

General Targeting

Shining light on a broad area activates opsin-infected cells.

Genetic Targeting

Uses viruses to target specific neurons within an area.

Projection Targeting

Targets neurons communicating via axons, enhancing specificity.

Viral Vector Targeting

Viruses are modified to carry opsin genes and deliver them to specific neurons.

Promoters

Ensure opsins are only expressed in desired cells.

Cre Recombinase

Cuts and integrates opsin DNA into neurons.

Ion Channels

Light-gated ion channels open when light activates opsins.

Behavioral Observation

Changes in behavior indicate neuron function.

Electrophysiological Recording

Direct measurement of electrical activity.

Calcium Imaging

Monitors calcium ions as indicators of neuronal activity.

2007 Study

Used to activate sleep-related neurons in mice.

Immune Response

Opsins from non-human sources (e.g., algae) are often attacked by the immune system.

LSFM

Uses a light sheet instead of an undermounted light to light the fluorophores in samples.

Benefits of LSFM

Samples do not have to be cut into thin slices and put on slides, so seeing deep into a sample is not much more work.

less

Is the risk of photobleaching more or less because you are lighting up less of the sample as a whole.

Optical Clearing

Method to make the sample clear so light can penetrate to deeper layers.

CLARITY

Infuse sample with hydrogel then wash with detergent to remove lipids.

Scale A2

Incubate in Scale clearing solutions to disrupt lipid structure and match RI of sample.

Excitation filter, Dichroic mirror (bounces off), Objective, Sample, Dichroic mirror (passes through), Emission filter, You.

How does the light get from lens to the sample and back to your eyes?

immunofluorescence microscopy

Uses antibodies conjugated with fluorophores (light-emitting molecules) to detect proteins in cells.

fluorophore

A chemical that absorbs and emits light, allowing visualization of cellular structures.

direct immunofluorescence

A fluorophore-conjugated antibody binds directly to the target protein

indirect immunofluorescence

primary antibody binds to the protein, followed by a fluorophore-conjugated secondary antibody for enhanced signal detection

Iterative Immunofluorescence Microscopy

method to image multiple targets in a single sample by sequentially staining, imaging, and removing antibodies

precise visualization of multiple components within a single sample

what are some advantages of iterative immunofluorescence?

illumina sequencing

next-generation sequencing (NGS) technology that uses sequencing by synthesis (SBS) to determine the order of nucleotides in DNA

sample preparation, library preparation, cluster generation, sequencing by synthesis, post-sequencing analysis

list the steps of illumina sequencing

identify tumor biomarkers, compare viruses, increasing crop yields

list a few applications of illumina sequencing

Hi-C

a technique used to characterize the 3D spatial organization of the genome in cell nuclei

interaction maps

what is the product of Hi-C?

not needed

genes packed further away in the nucleus because they are (needed/not needed).

clustered regularly interspaced short palindromic repeats

what does CRISPR stand for?

target DNA, cut DNA, repair the DNA

what are the 3 steps in CRISPR mechanisms?

gRNA

Guide RNA that directs Cas9 to target DNA.

Cas9 enzyme

bacterial enzyme which can make double stranded breaks to edit the genome

NHEJ and HDR

what are two cell responses to DNA breaks?