As far back as the 1950s, metal ion complexes have been used as imaging agents for detailed detection of dynamic intracellular processes, particularly in relation to disease pathogenesis. These complexes can selectively accumulate in organelles of interest through the ability of chemists to alter their chelating ligands. Recently, a set of these imaging agents, known as ReZolve and IraZolve dyes became commercially available and are transforming the way scientists visualize energy storage, signaling, cell metabolism, and major structural components of cellular membranes.
ReZolve and IraZolve dyes are derived from low-spin transition metal complexes (e.g., Re(I) tricarbonyl tetrazolo complexes and cyclometalated Ir(III) complexes). Emission from the low-spin metal ion complex results from charge transfer between the metal center and highly conjugated ligands. The charge transfer between metal and ligand produce long-lived fluorescent emission. These complexes are also color-tunable in that the emission of the charge transfer can be altered by incorporation of electron-withdrawing and electron-donating substituents within the metal-bound ligands that modulate the energy gap between the highest occupied and lowest unoccupied molecular orbitals.
These unique metal ion complex dyes exhibit high quantum yields, long-lived (millisecond) visible emission, are resistant to photobleaching, and have been developed to allow for live cell imaging. The clear advantage of this technology over small fluorescent molecules or fluorescently labelled antibodies is that it overcomes the limitations of photobleaching, short (nanosecond) emissive lifetimes, and the need for cell fixation.
|Localization||Polar Lipids and Lipid-Rich Compartments||Lipid Droplets and Endoplasmic Reticulum||Endoplasmic Reticulum||Mitochondria||User-Defined Luminescent Tag|
|Resistance to photobleaching||High||High||High||High||High|
|Ex/Em (nm)||UV or 405/550||405/600||UV or 405/570||405/600||UV or 405/570|
|Live Cells and Tissue Compatible||Yes||Yes||Yes||Yes||Yes|
|Fixed Cells and Tissues Compatible||Yes||Yes||Yes||Fixed Tissue Only, Not Fixed Cells||Yes|
|Fluorescence and Multiphoton Microscopy Compatible||Yes||Yes||Yes||Yes||Yes|
|Raman and Infrared Spectroscopy Compatible||Yes||No||Yes||No||Yes|
|Storage and Transport||Room Temp||Room Temp||Room Temp||Room Temp (4°C after reconstitution)||Room Temp|
Both ReZolve-L1™ and IraZolve-L1™ can be used to detect lipid droplets in lipid storage cells (Figure 1). ReZolve-L1™ has also been shown to be unique for labelling polar lipids and lipid-rich compartments. This includes cholesterol, sphingolipids, and phospholipids. These dyes have been used for effective monitoring of cellular lipid content and localization, which is particularly useful in the investigation of metabolic disease. Their spectral qualities are compatible with other commercially available fluorophores for multiplexed imaging.
Figure 1. ReZolve-L1™ detected in lipid droplets in adipocytes.
ReZolve-ER™ has rapid cell uptake and can be used for staining of the endoplasmic reticulum (ER), which allows observation of dynamic changes that occur in the ER (Figure 2). The use of this dye does not require wash steps, which decreases preparation time. This makes it ideal for time-dependent experiments or drug treatment assays where multiple wash steps can interrupt work flow. The stain is reversible and can be washed from cells after imaging. While IraZolve-L1™ localizes in lipid droplets, it can also accumulate in the ER.
Figure 2. ReZolve-ER™ in live and 4% paraformaldehyde-fixed prostate cells.
IraZolve-Mito™ is uniquely suitable for the detection of mitochondria in fixed and frozen tissue samples, as well as live cells (Figure 3). It has rapid cell uptake and allows for quick and easy detection of mitochondria in samples, without the need for antibody staining. Because it is amenable to long-term storage, IraZolve-Mito™ is ideal for animal trials where multiple samples are collected and stored for later analysis.
Figure 3. IraZolve-Mito™ on live H9C2 cells and images at 20x.
ReZolve-Alkyne™ can be used to track a compound of interest in cells or tissues by tagging azide functionalized molecules (Figure 4). This luminescent alkyne has a large Stokes shift, long emission lifetime, and is resistant to photobleaching. It can be conjugated to a range of chemicals and requires only simple or even no workup or purification of the product. Resulting products can be detected by fluorescence, infrared spectroscopy, Raman spectroscopy, X-ray fluorescence and inductively coupled plasma mass spectrometry, making it an ideal tool for both chemists and biologists.
Figure 4. ReZolve-Alkyne™ is suitable for copper-facilitated click reaction.
|Fluorophore concentration/dilution||There is a tendency to think more is better, but ReZolve products have an ideal concentration range. Using the dyes at too high a concentration can cause the dyes to precipitate, which then will not enter the cells.||All ReZolve dyes should be used at a final concentration of less than 50 µM, and for most, 10-20 µM is recommended. Verify the protocol and recommended concentration for each product.|
|Sample preparation||All ReZolve dyes work well in live cells. However, there are some limitations in fixed cell preparations. |
|Fixation at 2-4% PFA for 10-20 minutes is recommended to retain lipids, which enable localization.|
|Incubation time||Protocols for ReZolve dyes are only guidelines. Incubation times may need to be adjusted depending on the sample.||Longer or shorter incubation times may work better for your particular sample.|
|Mounting media or sample storage||Glycerol is a suitable mounting media but can dull the signal and extract the dye if the sample is stored for too long before imaging.||Water-based mounting media is recommended with ReZolve dyes. For best results, image the sample the same day as staining.|
|Imaging||ReZolve protocols provide a general recommendation for imaging. However, each microscopy system is different. Samples can vary greatly, and in some cases, emission/detection levels may seem faint.||Imaging can be improved by using lower capture speeds, or by leaving the detection open for longer to improve signal collection. The capture speed may be difficult to adjust when working with live samples, which can move during image collection.|
|Imaging settings||ReZolve dyes excite with a UV or 405 nm light source, unlike most blue-excited dyes, which have emission of 550-570 nm or 570-600 nm. Some older systems require filters for the excitation source, as well as the emitted light. If the filters are incorrect in epifluorescence microscopy, the excitation light can be blocked altogether.||Verify the emission filter is set appropriately for imaging FITC or Texas Red dyes. Verify filters (excitation source and emission light filters) are correct when using older systems. Confocal microscopy does not have this issue.|
Gillam, T.A., Sweetman, M.J., Bader, C.A., et al. Bright lights down under: Metal ion complexes turning the spotlight on metabolic processes at the cellular level. Coord. Chem. Rev. 375, 234-255 (2018).