An Introduction to Fluorescence Imaging

Imaging microscopic biological specimens demands greater resolution than can typically be attained via conventional microscopy. Consider a cell, which is predominantly composed of water and is subsequently transparent. Visible light-based microscopy will fail to provide much detail about cellular structures as most light on the range of 400—700 nanometres (nm) will simply transmit through. This makes it impossible to observe intracellular structures or sub-micro scale biological units like proteins using a conventional microscope.


What is Fluorescence Imaging?

Fluorescence imaging refers to a range of alternative microscopic and spectroscopic methods designed explicitly for visualizing small-scale biological processes and structures using much higher intensity light sources and fluorescent tagging. This resolves the problems of contrast and resolution in biological imaging, enabling researchers to localise extremely specific features at increasingly small scales. Differentiating one type of brain cell from another, for instance. Or, detecting genetic material within cells. So, how does fluorescence imaging work?


The Mechanism of Fluorescence

All types of fluorescence imaging exploit the mechanism of fluorescence to selectively excite molecules of interest. This occurs when a molecule tagged with a fluorescent label is probed with high-intensity light of the right wavelength. Electrons in the ground state transition to higher energy levels, but this excited state is intrinsically unstable. Gradually, electrons in the higher atomic orbital will emit energy and drop back into lower levels. This energy is observable as fluorescence.


Types of Fluorescence Imaging

Before the introduction of the first fluorescent dyes, researchers relied on brightfield microscopy to generate high contrast images of biomolecular samples. Modern fluorescence imaging systems offer radically improved specificity and 3D resolution, resulting in significant uptick of different imaging systems across the full spectrum of life sciences and bioengineering. A selection of common fluorescence imaging systems includes:


  • Fluorimetry
  • Fluorescent widefield microscopy
  • Total internal reflection microscopy (TIRF)
  • Fluorescence-lifetime imaging microscopy (FILM)
  • Fluorescence resonance energy transfer
  • Molecule localisation microscopy


Imaging System Configurations

Partnering fluorescent dyes with the right excitation wavelength is central to success in any fluorescence imaging system, and there are various methods of ensuring that low-noise fluorescent signals can be acquired directly. A fluorometer may use a multi-spectral light source with a wavelength range of 200—750 nm and a monochromator to selectively transmit light of finely-tuned, narrow wavebands. This is useful for measuring several components in a single scan as the diffraction grating can gradually cycle through multiple excitation wavelengths.


A faster and more efficient method of exciting specific fluorophores is to use laser light. Diodes and diode-pumped solid-state (DPSS) lasers provide much narrower linewidths thus greater monochromaticity, enabling fluorescence imaging much closer to the given excitation wavelength. Additionally, laser-based excitation enables fluorescence imaging to eschew expensive filtration optics used to fine-tune the excitation signal. This has numerous benefits including weight and size savings, as well as greater efficiency.