*2.1. Materials*

NIPAM was purchased from FIJIFILM Wako Pure Chemical Corporation (98%, Osaka, Japan, catalog no. 099-03695) and was purified by recrystallization from *n*-hexane. Cetyltrimethylammonium chloride (CTAC, 95%, catalog no. 087-06032), NaCl (99%, catalog no. 196-01671) and KCl (99.9%, catalog no. 164-13122) were purchased from FIJIFILM Wako Pure Chemical Corporation and were used without further purification. MBAM (99%, catalog no. 148326) and *<sup>N</sup>*,*N*,*N*',*N*'-tetramethylethylenediamine (TMEDA, 99%, catalog no. T22500) were purchased from Sigma-Aldrich Japan Inc. (Tokyo, Japan) and were used without further purification. D2O was obtained from Cambridge Isotope Laboratories (D, 99.9%, Tewksbury, US, catalog no. DLM-4-100). ADIP [31] and DBD-AA [32] were synthesized and purified, as previously reported. Water was purified using a Direct-Q 3 UV system (Merck Millipore, Burlington, MA, USA). Dialysis membranes (MEMBRA-CEL MD34, Molecular Weight Cut Off: 14,000; Pore size: 50 Å) were purchased from Viskase Companies Inc. (Willowbrook, IL, USA).

35-mm Glass dish was purchased from Iwaki/AGC Techno Glass (Shizuoka, Japan, catalog no. 3960-035). Phenol red-free DMEM (Dulbecco's modified Eagle's medium, catalog no. 21063-029), FBS (Fetal Bovine Serum, Hyclone, catalog no. SH30910.03), 0.5% Trypsin-EDTA (ethylenediaminetetraacetic acid, catalog no. 15400054) and MitoTracker Deep Red FM (catalog no. M22426) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DMEM (catalog no. 08458-45), Na2HPO4·12H2O (98%, catalog no. 31722-45) and KH2PO4 (99.5%, catalog no. 28721-55) were purchased from Nacalai Tesque (Kyoto, Japan). HeLa (human epithelial carcinoma) cells were obtained from ATCC (catalog no. CCL-2, Manassas, USA).

#### *2.2. Preparation of Cationic Nanogels (NANOGEL-1 and NANOGEL-2) and Cationic Fluorescent Nanogel Thermometers (NANOGEL-3~6).*

NIPAM, DBD-AA, MBAM, TMEDA and/or CTAC were dissolved in 19 mL of water (for the final concentration of each compound in a reaction mixture, see Table 1). Dry argon gas was bubbled through the solution at 70 ◦C for 30 min to remove the dissolved oxygen. ADIP [31] in water (1 mL) was added to initiate polymerization, and the mixture was stirred using a rod with a paddle at 250 rpm and 70 ◦C for 1 h under an argon atmosphere. The mixture was then poured into 400 mL of water, and the nanogels were precipitated by a salting-out technique. After purification by dialysis for at least one week, the nanogel dispersions were freeze-dried. The purity of NANOGEL-**1**~**6** was confirmed by 1H-NMR measurements with a Bruker AVANCE 400 spectrometer (Billerica, MA, USA) in D2O (Figure S1). The yields are indicated in Table 1.


**Table 1.** Preparation of cationic nanogels (NANOGEL-**1** and NANOGEL-**2**) and cationic fluorescent nanogel thermometers (NANOGEL-**3**~**6**).

#### *2.3. Characterization of NANOGEL-1~6*

TEM images were obtained with a Hitachi H-7100 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan). A drop of the nanogel solution in ethanol or water (0.01 w/v%, 5 μL) was placed on a formvar-coated copper grid. The specimen was air-dried at room temperature and then examined at an accelerating voltage of 75 kV. The hydrodynamic diameter and zeta potential were measured with a Malvern Instruments Zetasizer Nano ZS (currently Malvern Panalytical, Malvern, UK). The samples were equilibrated for 10 min at each temperature. The amount of fluorescent DBD-AA units in NANOGEL-**3**~**6** was estimated from the comparison of the absorbance of their methanol solution with that of *<sup>N</sup>*,2-dimethyl-*N*-(2-{methyl[7-(dimethylsulfamoyl)-2,1,3-benzoxadiazol-4-yl]amino}ethyl)propenamide (ε = 11,000 M−<sup>1</sup> cm<sup>−</sup><sup>1</sup> at 444 nm) as a model compound [33]. The fluorescence spectra of NANOGEL-**3**~**6** were recorded in water and a 150 mM KCl solution at various temperatures using a JASCO FP-6500 spectrofluorometer (Tokyo, Japan) with a Hamamatsu R-7029 optional photomultiplier tube (Hamamatsu, Japan, operating range, 200–850 nm). The sample temperature was controlled using a JASCO ETC-273T temperature controller (Tokyo, Japan).

#### *2.4. Introduction of Cationic Fluorescent Nanogel Thermometers into HeLa Cells*

The HeLa cells were cultured on a 35-mm glass dish in high-glucose DMEM supplemented with FBS at 37 ◦C with 5% CO2. The cationic fluorescent nanogel thermometers were introduced into the HeLa cells by two different methods: a standard method using cells adhered to a glass-bottom dish and a modified method using suspended cells. For the standard method, DMEM was removed from a glass-bottom dish containing HeLa cells at 30–50% confluency, and the cells were rinsed with 1 mL of 1×PBS (phosphate buffered saline, 10×PBS containing 28.9 g of Na2HPO4·12H2O, 2.0 g of KH2PO4, 80.0 g of NaCl and 2.0 g of KCl in a 1 liter solution). Then, PBS was replaced by 1 mL of cationic fluorescent nanogel thermometers in a 5 w/v% glucose solution (0.05 w/v%, 10 μL of a 5 w/v% stock solution in water diluted in 990 μL of a 5 w/v% glucose solution). A 5 w/v% stock solution in water was prepared and incubated at 4 ◦C at least overnight before the full solvation of nanogels. The dish was incubated at 25 or 37 ◦C for 5, 10 or 20 min without CO2 supply. After incubation, the nanogel solution was removed, and the cells were rinsed with 1 mL of 1×PBS three times. Two milliliters of phenol red-free DMEM was added to the dish before imaging.

For the modified method using suspended cells, HeLa cells cultured in 100 mm culture dishes were rinsed with 1 mL of 1×PBS, then treated with 0.5 mL of a 0.05 w/v% trypsin-EDTA-1×PBS solution and incubated at 37 ◦C for 3–5 min. The detached cells were suspended in 9 mL of DMEM. One milliliter of the cell suspension was transferred to 1.5 mL tubes and centrifuged at 1200 rpm at 4 ◦C for 1 min. The collected cells were rinsed twice with 1 mL of 1×PBS, and then suspended in 1 mL of cationic fluorescent nanogel thermometers ina5w/v% glucose solution. After incubation at 25 ◦C for 20 min without CO2 supply, the cells were again collected by centrifugation at 1200 rpm at 4 ◦C for 1 min and rinsed with 1 mL of 1×PBS three times before being suspended in 10 mL of DMEM. Two milliliters mL of cell suspension was added to a 35-mm glass-bottom dish and incubated at 37 ◦C with 5 % CO2 for one or two nights before observation.

#### *2.5. Fluorescence Imaging of HeLa Cells*

Confocal fluorescence imaging was performed using a TCS-SP5 laser scanning confocal microscope equipped with an HCX PL APO Ibd.BL 63 × 1.4 N.A. oil objective (Leica Microsystems, Wetzlar, Germany). Cells loaded with cationic fluorescent nanogel thermometers were excited by a 458 nm argon laser, then the fluorescence images were acquired through bandpass 500–700 nm in a 1024 × 1024 pixel format, with zoom factors ranging from 1 to 10 and a scanning speed of 400 Hz. The contrast and brightness of the fluorescence images were enhanced using ImageJ with a constant signal intensity ratio. The incorporation efficiencies (%) of NANOGEL-**6** were determined using Equation (1),

$$\begin{aligned} \text{Incoraporation efficiency (\%)} &= \text{number of cells containing NANDOGE-6} \\ &\quad / \text{number of cells} \times 100, \end{aligned} \tag{1}$$

in which the total cell number was 183–401, and the cells that showed a fluorescence intensity higher than the threshold (equal to the maximum autofluorescence intensity) were counted as the "cells containing NANOGEL-**6**". For the co-visualization of NANOGEL-**6** and mitochondria, the HeLa cells were stained with 50 nM MitoTracker Deep Red FM (MitoTracker DR) in phenol red-free DMEM for 5 min at room temperature and then treated with NANOGEL-**6** by a standard method using adhered cells. A 458 nm argon laser was used to excite NANOGEL-**6**, and a 633 nm HeNe laser was used to excite MitoTracker DR. The fluorescence of NANOGEL-**6** was collected through bandpass 500–600 nm, and the fluorescence of MitoTracker DR was collected through bandpass 645–730 nm.
