Category Archives: Physics

Understanding the Diffraction Limit in Microscopy


Basic light microscopy can only resolve objects that are larger than 100 nm which means that while it can visualize animal cells (~10,000nm), organelles and bacteria (~1,000nm), it cannot visualize viruses (<100nm), proteins (<10nm) or small molecules(~1nm) (see post summarizing Biological Scales). This limitation is known as the “diffraction limit” and is caused by the fact light only interacts differently with objects separated by more than one wavelength (λ). Intuitively, its helpful to think of each of these wavelengths as “a minimum pixel size” for a computer image where: infrared light (λ ~ 10.0μm) has pixels 100 times larger than ultraviolet light (λ ~ 0.1μm).

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The “Spectrum” of Microscopic and Spectroscopic Techniques


Electromagnetic (EM) radiation is our main source of information about the world (i.e. “seeing is believing”). Most scientific techniques rely on some form of imaging/visualization (“microscopy”) and/or measurement of energy absorption or emission (“spectroscopy”) or both (for instance: fluorescence microscopy). In the figure above we outline the spectrum of electromagnetic radiation from x-rays to microwaves and the different scientific techniques that each type of radiation supports.

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Why are Emission and Excitation Spectra Mirror Images?

09 Emission-Excitation Spectra

In a follow-up to our introduction to fluorescence, we wanted to discuss a somewhat confusing(at least for us) detail of fluorescence spectroscopy: the fact that emission spectra and excitation spectra of the same fluorophore are mirror images of each other. It wasn’t until we drew out the diagram pictured above that we truly “got it.”

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Introduction to Fluorescence

07 Fluorescence Introduction

Fluorescence (i.e. the emission of light from a electronically excited substance) is one of the most utilized physical phenomena in chemistry and biology. Though humans have been aware of fluorescence for thousands of years (e.g. fireflies), it wasn’t until the discovery of quinine in 1845 that we really started to understand its chemical basis(see figure above). Interestingly, during World War II, the study of quinine’s anti-malarial properties led to the development of the first spectrofluorometers which enabled true quantitative study of fluorescence. Finally, in the 1980-1990’s, new tools in synthetic chemistry and molecular biology allowed rational engineering of chemical fluorophores into a critical class of probes in biophysics(microscopy), molecular biology(sequencing), cell-biology(flow cytometry) and even anatomy (histology)).

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Intuiting Biological Scales using Human Scales

Figure 1. Human Scale Key: Aircraft Carrier (200m), 3-story House (20m), Human (2m), Liver/Gerbil (200mm), Lymph Node/Mosquito (20mm), Skin/Flee (2mm), Hair thickness (200um)
Biological Scale Key: Flee (2mm), Amoeba (200um), Eukaryotic Cell (20um), Mitochondria/Bacteria (2um), Centriol/Large Virus (200nm), Cell Membrane/Small Virus (20nm), dsDNA thickness (2nm)

The length scale of molecular and cellular biology (Figure 1 Biological Scales) covers approximately 5 orders of magnitude, with sizes ranging from 1 nanometer (approximate size of small molecules) to 100 micrometers (approximate size of the largest cells). Unfortunately, it can be difficult to intuit these relative sizes because we cannot directly observe these scales.

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