Heat application in live cell imaging

Abstract

Thermal heating of biological samples allows to reversibly manipulate cellular processes with high temporal and spatial resolution. Manifold heating techniques in combination with live-cell imaging were developed, commonly tailored to customized applications. They include Peltier elements and microfluidics for homogenous sample heating as well as infrared lasers and radiation absorption by nanostructures for spot heating. A prerequisite of all techniques is that the induced temperature changes are measured precisely which can be the main challenge considering subcellular structures or multicellular organisms as target regions. This article discusses heating and temperature sensing techniques for live-cell imaging, leading to future applications in cell biology.

Keywords: fluorescence microscopy; live‐cell imaging; microheating; thermometry.

Fig. 1

Thermal heating in live‐cell imaging to induce biological processes with high spatiotemporal resolution. (A) Optothermal sensors allow to monitor the temperature in the cell with microscopic resolution. Small organic dyes track different cellular compartments by temperature‐dependent fluorescence. Polymeric temperature sensors rely on conformational changes. Nanomaterials allow to engineer optical thermometers with versatile optical properties. Fluorescent proteins can be readily expressed in cells. (B) Direct heating with mid‐IR radiation allows label‐free detection of (bio)molecules. (C) Direct heating of cells by nIR radiation is used to induce cytoplasmic flow, inactivate temperature‐sensitive (ts) proteins, probe protein stability and sample interaction kinetics inside cells. (D) Indirect heating via excitation of nanostructures using optical, magnetical or ultrasound excitation. Examples include the optical excitation of nanoparticles to regulate transcriptional activity utilizing a heat shock promoter (left), magnetic excitation of magnetic particles (MP) controlling calcium signaling (middle), and ultrasound excitation of nanoparticles for controlled cell death in cancer treatment (right).

Fig. 2

Optical thermometers to determine intracellular temperatures. (A) A rosamine‐based fluorescent dye colocalized with mitochondria serving as an organelle specific temperature probe. Aluminum particles were heated with a nIR laser in proximity to the cells. The images show its application in different cell cultures with a comparable temperature sensitivity of 2.5–2.8% per °C. Scale bar = 20 μm. Adapted and reprinted from [31] with permission. (B) Genetically encoded temperature sensor based on the fluorescent proteins mVenus (mV) and the mTurquoise2 (mT) separated by elastin‐like polypeptide linker (ELP). Upon temperature increase the sensor self‐associated, allowing a ratiometric readout of the temperature (mV/mT). The study revealed a temperature difference between the cytosol and the nucleus in HeLa cells. Scale bar = 20 μm. Adapted and reprinted from [34] with permission.

Fig. 3

Heat application by direct excitation using IR lasers. (A) FLUCS was used to induce a cytoplasmic flow by clockwise movement of an IR laser in a C. elegans zygote. The pattern of the GFP‐tagged partition‐defective protein (PAR) was redistributed 420 s after the induced flow. Scale bar = 10 μm. Reprinted from [53] with permission. (B) FLIRT allowed reversible inactivation of temperature‐sensitive proteins (myosin‐II) in C. elegans embryos during cell division using spatially controlled IR laser heating. Scale bar = 10 μm. Reprinted from [58] with permission. (C) Thermal stability and unfolding of superoxide dismutase 1 (SOD1) in the endoplasmatic reticulum investigated by FReI. SOD1 was labeled C‐ and N‐terminally with a FRET pair to track unfolding. A temperature profile was applied to the cell to induce stepwise unfolding. Kinetic unfolding amplitudes were used to determine the melting temperature (T _m) as a measure of folding stability. Scale bar = 20 μm. Reprinted from [74] with permission. (D) TOOL microscopy was used to study DNA hybridization by an oscillating temperature. Pixel‐by‐pixel analysis revealed millisecond kinetics within single cells. Scale bar = 10 μm. Reprinted from [73] with permission.

Fig. 4

Indirect heat application by opto‐ and magnetothermal nanoparticles. (A) Calcium release from endosomes was induced by exciting plasmonic nanoparticles close to endosomes. Ca²⁺ release and distribution in the cell was monitored by Fluo‐4AM. Scale bar = 20 μm. Reprinted from [78] with permission. (B) Gene expression was triggered by activation of thermal gene switches which are based on the promoter of the heat shock protein HSP70B'. Pulsed excitation of gold nanorods enabled long‐term control of transcriptional activity in engineered T‐cells in mice. Reprinted from [79] with permission. (C) Magnetic nanoheaters were used to induce local hyperthermia and therefore control the cell death of particular cells. Reprinted from [89] with permission.