**1. Introduction**

Fluorescent molecular thermometers change their fluorescence properties (e.g., fluorescence intensity, fluorescence quantum yield, fluorescence lifetime and maximum emission wavelength) with temperature [1–5]. Fluorescent polymeric thermometers based on the combination of a thermoresponsive polymer and an environment-sensitive fluorophore [6–12] are among the most sensitive fluorescent molecular thermometers. Because of their function at the molecular level in aqueous media, these sensitive fluorescent polymeric thermometers have been applied for the thermometry of small subjects, such as microfluids [13,14] and even single living cells [15–25]. Fluorescent polymeric thermometers are classified into two categories of morphology: fluorescent nanogel thermometer with crosslinking units to construct a nano-scaled particle [8,15] and fluorescent linear polymeric thermometer without crosslinking units [6,7,16]. In general, the former is more robust and less interactive with external molecules and ions, whereas the latter is capable of thermometry with a higher spatial resolution, due to its diffusivity.

In 2009, we performed the first intracellular thermometry of live mammalian COS7 (African green monkey kidney) cells using a fluorescent nanogel thermometer that was negatively charged, due to the radical initiator ammonium persulfate (APS) used in its preparation [15]. For intracellular thermometry, the microinjection technique was required for the introduction of anionic fluorescent nanogel thermometers into live COS7 cells. If the ability to spontaneously enter live cells is expected, a

fluorescent nanogel thermometer should be positively charged based on the established concept that polycationic structures e fficiently support the spontaneous entry of molecules into living cells [26–30]. However, the preparation of positively charged fluorescent nanogel thermometers has not been easily realized because of the lack of cationic radical initiators.

In a recent communication published in 2018 [31], we reported the use of the first cationic radical initiator, 2,2'-azobis-[2-(1,3-dimethyl-4,5-dihydro-1 *H*-imidazol-3-ium-2-yl)]propane triflate (ADIP), to prepare cationic nanogels, including a cationic fluorescent nanogel thermometer (Figure 1). In the present article, we describe a detailed experimental procedure to prepare cationic NIPAM nanogels and NIPAM-based cationic fluorescent nanogel thermometers using ADIP and present the comprehensive physical and photophysical properties of these cationic nanogels (i.e., size, zeta potential and/or temperature-dependent fluorescence properties) and the functions of the cationic fluorescent nanogel thermometers in intracellular applications (i.e., ability to enter live cells, distribution in cells, cytotoxicity and response to temperature variation). These experimental protocols and new functional data will complement our earlier communication [31].

**Figure 1.** Cationic fluorescent nanogel thermometer prepared with cationic radical initiator ADIP (2,2'-azobis-[2-(1,3-dimethyl-4,5-dihydro-1 *H*-imidazol-3-ium-2-yl)]propane triflate). NIPAM: *N*-isopropylacrylamide, DBD-AA: *N*-{2-(7- *<sup>N</sup>*,*<sup>N</sup>*-dimethylaminosulfonyl-2,1,3-benzoxadiazol-4- yl)methylamino}ethyl-*N*-methylacrylamide, MBAM: *<sup>N</sup>*,*<sup>N</sup>*'-methylenebisacrylamide.

#### **2. Materials and Methods**

Bulletized procedures corresponding to Sections 2.2 and 2.4 were described in Supplementary Materials.
