Microscopy – 5 Reasons Coverslips Are Important For High-Quality Imaging

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

2020 was a difficult year for many, with their own research being interrupted- either by lab shutdowns or recruitment into the race against COVID-19. Despite the challenges, scientists have continued to be creative and have pushed the boundaries of what is possible. These are the techniques and technologies that every microscopist was envious of in 2020. Spatially Resolved Transcriptomics Nature Methods declared that spatially resolved transcriptomics was the 2020 method of the year. These are a  group of methods that combine gene expression with their physical location. Single-cell RNA sequencing (scRNAseq) was originally developed for cells that had been dissociated…

As biologists, we study the process of life, however, it’s intricacies cannot be captured by a snapshot in time. Generally, the easiest imaging experiments are those where the samples are stained, fixed, and imaged within a few days of procurement, but that too doesn’t capture the dynamic processes common in cells and organisms. Live cell imaging when combined with reporters serves as a powerful tool to provide solid imaging data. Cameleon —one of the first reporters— was developed in 1997 in Roger Tsien’s lab.  Cameleon is a green fluorescent protein (GFP) that undergoes a conformational change in the presence of…

As scientists, we need to perform image analysis after we’ve acquired images in the microscope, otherwise, we have just a pretty picture and not data. The vocabulary for image processing and analysis can be a little intimidating to those new to the field. Therefore, in this blog, I’m going to break down 7 terms that are key when post-processing of images. 1. RGB Image Images acquired during microscopy can be grouped into two main categories. Either monochrome (that can be multichannel) or “RGB.” RGB stands for red, green, blue – the primary colors of light. The cameras in our phones…

In an ideal world, we would be able to use fluorophores that don’t have any overlap in emission spectra and autofluorescence wouldn’t obscure your signal. Unfortunately, we don’t live in such a world and often have to use two closely related dyes – or contend with fluorescent molecules that are innately part of our sample. Fluorescent molecules include chlorophyll, collagen, NADPH, and vitamin A.  One example that I recently encountered was developing a new probe for lipids. The reviewers requested a direct comparison of the new dye to Nile Red in the same sample. Both dyes would localize to the…

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…