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. Key Takeaways Fluorescence imaging uses light to make specific molecules ‘glow’ so they can be seen clearly. The process uses high-intensity light sources and a range of microscopic and spectroscopic methods Scientists attach fluorescent labels to the molecules they want to study. When high‑intensity light of a specific wavelength illuminates these labeled molecules, their electrons absorb the energy and transition from a ground state to an excited state. Because this excited state is unstable, the electrons quickly return to a lower‑energy state and release the excess energy as light (fluorescence) Laser light provides the most efficient method of exciting specific fluorophores, with diodes and diode-pumped solid-state (DPSS) lasers providing narrower linewidths There are different types of fluorescence imaging, including fluorimetry, fluorescent widefield microscopy, total internal reflection microscopy (TIRF), and fluorescence-liftime imaging microscopy (FILM) Fluorescence imaging is used in life sciences, bioengineering, and biological imaging in applications such as differentiating cells or detecting genetic material The benefits of modern fluorescence imaging systems include improved specificity and enhanced 3D resolution Contents What is fluorescence imaging? The mechanism of fluorescence Types of fluorescence imaging Fluorescence imaging system configurations About Novanta Precision Manufacturing 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 Fluorescence 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. About Novanta Precision Manufacturing Who we are is inevitably embedded in what we do, our innovations, and the people that make it happen. Our core strength is delivering market-leading solutions for our customers, but also maintaining deep and long-lasting customer relationships. We do this through our global application sales force, and quality-focused manufacturing expertise. It is thanks to what we accomplished in our past and our continuous work towards innovation, that makes us who we are today. Novanta is a trusted technology partner to medical and industrial OEMs (original equipment manufacturers). Additionally, Novanta holds deep proprietary expertise in photonics, vision and precision motion technologies. We engineer mission-critical core components and subsystems that deliver extreme precision and performance. This enabling our customers to improve productivity, to achieve breakthrough performance and to enhance people’s lives. Building a high-performing culture enables us to achieve our growth goals. It starts with cohesive teams that engage and align around our vision and strategy. Those who live our values and drive performance through the Novanta Growth System. This is a common set of tools and processes for continuous improvement. Our highly engineered component and sub-system solutions, and deep expertise in advanced photonics, vision and precision motion make us the global technology partner of choice for medical and advanced industrial OEMs. Learn more about Novanta by contacting us here.
